Publications and other reference materials referred to herein are incorporated herein by reference and are numerically referenced in the following text and respectively grouped in the appended Bibliography which immediately precedes the claims.
The present invention is directed to novel methods of detecting and locating specific sequences in double stranded DNA using nucleoside oligomers which are capable of specifically complexing with a selected double stranded DNA Structure to give a triple helix structure.
Formation of triple helix structures by homopyrimidine oligodeoxyribonucleotides binding to polypurine tracts in double stranded DNA by Hoogsteen hydrogen bonding has been reported. (See, e.g. (1) and (2)). The homopyrimidine oligonucleotides were found to recognize extended purine sequences in the major groove of double helical DNA via triple helix formation. Specificity was found to be imparted by Hoogsteen base pairing between the homopyrimidine oligonucleotide and the purine strand of the Watson-Crick duplex DNA. Triple helical complexes containing cytosine and thymidine on the Hoogsteen (or third) strand have been found to be stable in acidic to neutral solutions, respectively, but have been found to dissociate on increasing pH. Incorporation of modified bases of T, such as 5-bromo-uracil and C, such as 5-methylcytosine, into the Hoogsteen strand has been found to increase stability of the triple helix over a higher pH range. In order for cytosine (C) to participate in the Hoogsteen-type pairing, a hydrogen must be available on the 3-N of the pyrimidine ring for hydrogen bonding. Accordingly, in some circumstances, C may be protonated at N-3.
DNA exhibits a wide range of polymorphic conformations, such conformations may be essential for biological processes. Modulation of signal transduction by sequence-specific protein-DNA binding and molecular interactions such as transcription, translation, and replication, are believed to be dependent upon DNA conformation. (3)
It is exciting to consider the possibility of developing therapeutic agents which bind to critical regions of the genome and selectively inhibit the function, replication, and survival of abnormal cells. (4) The design and development of sequence-specific DNA binding molecules has been pursued by various laboratories and has far-reaching implications for the diagnosis and treatment of diseases involving foreign genetic materials (such as viruses) or alterations in genomic DNA (such as cancer).
Nuclease-resistant nonionic oligodeoxynucleotides (ODN) consisting of a methylphosphonate (MP) backbone have been studied in vitro and in vivo as potential anticancer, antiviral and antibacterial agents. (5) The 5'-3' linked internucleotide bonds of these analogs closely approximate the conformation of nucleic acid phosphodiester bonds. The phosphate backbone is rendered neutral by methyl substitution of one anionic phosphoryl oxygen; decreasing inter- and intrastrand repulsion due to the charged phosphate groups. (5) Analogs with MP backbone can penetrate living cells and have been shown to inhibit mRNA translation in globin synthesis and vesicular stomatitis viral protein synthesis, and inhibit splicing of pre-mRNA in inhibition of HSV replication. Mechanisms of action for inhibition by the nonionic analogs include formation of stable complexes with complementary RNA and or DNA.
Nonionic oligonucleoside alkyl- and aryl-phosphonate analogs complementary to a selected single stranded foreign nucleic acid sequence can selectively inhibit the expression or function or expression of that particular nucleic acid without disturbing the function or expression of other nucleic acids present in the cell, by binding to or interfering with that nucleic acid. (See, e.g. U.S. Pat. Nos. 4,469,865 and 4,511,713). The use of complementary nuclease-resistant nonionic oligonucleoside methylphosphonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including Herpes simplex virus-1 is disclosed in U.S. Pat. No. 4,757,055.
The use of anti-sense oligonucleotides or phosphorothioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral mRNA into protein has been proposed. The anti-sense constructs can bind to viral mRNA and were thought to obstruct the cells ribosomes from moving along the mRNA and thereby halting the translation of mRNA into protein, a process called "translation arrest" or "ribosomal-hybridization arrest." (6)
The inhibition of infection of cells by HTLV-III by administration of oligonucleotides complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and or expression is disclosed in U.S. Pat. No. 4,806,463. The oligonucleotides were found to affect viral replication and or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins p15 and p24 (gene expression).
The ability of some antisense oligodeoxynucleotides containing internucleoside methylphosphonate linkages to inhibit HIV-induced syncytium formation and expression has been studied. (7)
Psoralen-derivatized oligonucleoside methylphosphonates have been reported capable of cross-linking either coding or noncoding single stranded DNA; however, double stranded DNA was not cross-linked. (28)